Innovative approaches for screening TRPV1 antagonists using noxious heat as a stimulus for opening the channel

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel that is expressed heterologously and stimulated by the same stimuli that excite C and Ad fiber nociceptors in vivo such as capsaicin and noxious heat (>43°C) [Caterina MJ et al, Nature 1997; 389:816-824].  Reduction of the activity of the channel is therefore proposed to reduce inflammatory pain [Szallasi A and Blumberg PM, Pharmacol. Rev. 1999; 51: 159-212].  However, there is a risk associated with this approach; raised core body temperature, as reported by Amgen in a recent clinical trial of AMG517 [Wong GY and Gavva NR, Brain Res. Rev. 2009; 60: 267-277].  Therefore, development of robust in vitro assays that use heat activation of the TRPV1 receptor is essential to identify antagonists of the channel that do not cause hyperthermia.

Existing microplate readers for ion channel cell based assays do not control temperature within individual wells, so we have adopted an evolving strategy to successfully address this problem.  Firstly, we developed an unconventional calcium influx assay with fluorescence readout using an RT-PCR machine capable of rapidly and accurately ramping to target temperature within each well.  Secondly, we established a high-throughput calcium mobilisation assay using a custom-built temperature control unit which was fitted into the FLIPRÔ (Molecular Devices).  And thirdly, we have used the Patchliner platform (Nanion) to derive high quality electrophysiological data. 

The successful outcome of these innovative approaches to TRPV1 antagonist assay development has increased our ability to identify compounds with reduced risk of raising core body temperature.

M. PAPAKOSTA¹, J. DECOCK², R. LANE¹, A. ALDERTON¹, A. DUNLOP³, A. HAYTHORNTHWAITE⁴, R. WARD⁵, R. RUSSELL¹

¹Primary Pharmacology Group, Pfizer Ltd., UK; ²School of Biomedical & Health Sciences, King’s College London, UK; ³Data Support & Management, Pfizer Ltd., UK; ⁴Nanion Technologies, Germany; ⁵Pain RU, Pfizer Ltd., UK